My positive tissue control contained certain areas with dark brown staining and then my negative tissue control contained no brown areas, which is what I should be seeing. However in both samples I see some black spotting. I am trying to figure out why that is, the DAB i'm using is expired so I think that might be why but thought I'd post on here and see if there would be any other explanation?
Thanks!
Hi, I have experienced brown spots not black ones in IHC. In your case, i think it could be expired DAB deposits or formalin pigments if your specimens are over-fixed.
I am testing the DAB right now so hopefully that's all it is. I'm working with mice tissues; brain, kidney, liver, spleen and tumour. It's spotting on all of them.
Formalin pigment is not caused by prolonged fixation, but by acid formalin fixation without buffering. Do you use neutral buffered formalin?
Are the black spots only in the tissue or also over the glass slide?
They are only on the tissue. The fixation process is performed by the hospital's lab for all the researchers so I doupt it's an issue there. I double checked with my colleagues and they've never encountered this problem.
I also guess it's the DAB. Especially if the spots are spread without a pattern.
If the spots would be at the same place in every consecutive section, one must think about a reason that lies in the tissue itself (necrosis, crushed areas).
Hmm, I just did the same samples without DAB and it's still giving me the black spotting. I looked at a closer magnification and it seems likes it's just alot of very tiny bubbles.. I guess when I put the coverslips on? But i don't get why it's only where the tissue is at or how I can avoid this..
To avoid tiny bubbles thorough dehydration is needed. After staining 2x 96%, 2x 100%, 2x xylen. Each 30-60 sek. with moving the slides until the surface looks smooth.
Possibly you have initially fixed your samples by 4% formalin and you have not transferred your samples to a new batch of 4% formalin after 24 hour. Please either use a less concentrated formalin for example 1-2% or substitute your old formalin with a new batch after 24 hour. After this step you can keep your samples in this formalin as long as you wish
Figured it out! I was letting my slides dry after the xylene step during dehydration. When I added the coverslips immedietly after taking them out of the xylene I got no bubbles on my slides. Thanks for all your answers!
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